The present invention relates to methods for derivatising proteins, in which a denaturant is included in the reaction mixture, to achieve high degrees of substitution. The method is of particular applicability to derivatisation methods involving reaction of an aldehyde reagent with epsilon amino groups of non-terminal lysyl units of proteins. Novel derivatised protein compounds have high degrees of substitution of polysialic acid chains.
In our earlier application number WO-A-92/22331 we describe methods in which polysaccharides, especially polysialic acids, are used to derivatise drug delivery systems or proteins to increase the circulation time, decrease the immunogenicity and/or increase the stability in vivo of the substrates. There is no worked example of a derivatisation reaction with any protein. Roy et al in J. Chem. Soc. Chem. Comm. 1993, 264-265 describe derivatisation by Michael addition to primary amine groups on proteins of an acrylic-functional sialic acid. The reaction is carried out in the presence of ammonium bicarbonate, which is not generally described as a denaturant.
It is well known that sodium dodecyl sulphate, at the concentration of 0.01M, has an effect on the three dimensional conformation of proteins in aqueous compositions. For instance, Prakash et al in Int. J. Peptide Protein Res. (1980) 15, 305-313, show using circular dichroic spectra that SDS induces more α helical structure in the α-globulin of Sesamum indicum L (a sesame seed). Visser et al, in Biochemistry (1971) 10(5) 743-752 use various optical characteristics of elastase, and other enzymes to determine changes in conformation in the presence of SDS. The protein concentrations in the solutions varied from 0.01 to 0.1%, whilst the SDS concentration was in the range 0.2 to 2% by weight. SDS was shown to inhibit irreversibly the activity of several enzymes under these conditions.
It is known to reduce disulphide bridges between two cysteine units of a protein in the presence of urea which facilitates unfolding of the protein and increase the accessibility of the disulphide groups to mercaptoethanol reducing agent.
As far as the present inventor is aware, anionic amphiphilic compounds, such as SDS, have not been used to affect the conformation of proteins in solution during aqueous derivatisation procedures.